Project Summary Despite long held beliefs that the human erythrocyte is an ?inert bag of hemoglobin?, many studies now reveal that the human red blood cell (RBC) is sensitively regulated by a variety of stimuli, including O2 pressure, mechanical deformation, multiple hormones and oxidative stress. We have shown recently that stimuli that promote tyrosine phosphorylation of the major erythrocyte membrane protein, band 3 (a.k.a. SLC4A1, anion exchanger, AE1), also induce erythrocyte membrane destabilization, leading in many cases to membrane vesiculation and cell fragmentation. More detailed exploration of the mechanism underlying this membrane destabilization has revealed that phosphorylation of the cytoplasmic domain of band 3 induces its intramolecular docking with a highly atypical ?SH2-like structure? within the membrane-spanning domain of band 3, resulting in rupture of the band 3-ankyrin bridge that connects the membrane to its cytoskeleton (and thereby promoting the aforementioned membrane destabilization). While this observation was at first perplexing, upon subsequent discovery that tyrosine phosphorylation of band 3 does not occur in healthy RBCs but is prominent in both sickle cells and malaria parasite-infected RBCs, we wondered whether the phosphorylation-induced membrane destabilization might contribute to development of these diseases. Follow-on studies revealed that inhibitors of band 3 tyrosine phosphorylation also inhibited: 1) the membrane weakening required for egress of malaria parasites from their RBC hosts, resulting in termination of the parasitemia, and 2) the membrane destabilization responsible for the intravascular hemolysis and release of membrane microparticles that are reported to trigger vaso-occlusive events in sickle cell patients. Motivated by these and other confirming observations, we have proposed in Aim 1 to characterize the phosphorylation pathways that lead to band 3 tyrosine phosphorylation and membrane destabilization in both sickle cells and malaria-infected RBCs. In Aim 2 we will use this information to design and evaluate inhibitors of these pathway(s) for possible use as therapeutics for treatment of the two pathologies. In Aim 3 we will characterize the phosphotyrosine binding properties of homologous ?SH2-like structures? that we have found in 47 other solute transporters of highly diverse functions and different evolutionary families. Because most of these transporters have significant pathologies associated with their malfunctions (e.g. the serotonin, dopamine, glutamate, glucose, and nucleoside transporters, etc.), elucidation of their mechanisms of regulation by tyrosine phosphorylation should reveal new approaches for treatment of their associated diseases. Thus, collectively, the information generated by the proposed studies will not only yield information that could lead to treatments for malaria and sickle cell disease, but also for a variety of other human maladies caused by the malfunction of important solute transporters.